Targeting Inhibition Of GRP75 Enhances The Sensitivity Of Hepatoma Cells To HSP90 Inhibitor 17-AAG

Part Ⅰ Targeting GRP75improves HSP90inhibitor efficacy by enhancing p53-mediated apoptosis in hepatocellular carcinomaHepatocellular carcinoma (HCC) is one of the most common malignant tumors and the second leading cause of cancer death worldwide, every year, there are more than110thousands people died of HCC. Although surgical resection and liver transplantation can be curative in early-diagnosed cases or in patients with localized tumors, approximately80%of patients with advanced liver cancer must largely rely on conventional chemotherapies, which have limited effectiveness due to the development of drug resistance and undesirable side effects. For this reason, new and effective therapeutic strategies for HCC are urgently needed.It was found that the expression of Heat Shock Proteins (HSPs) in cancer tissues is higher than that in non-tumor tissues, some of HSPs can be used as a marker for dignosis. For these reasons, to evidence the relationship between the HSPs and tumorigenesis and development of cancer will important for dignosis, therapy and prognosis of cancer. We use the mRNA array data from GEO dadabase, tissue array of HCC to analyse the potencial relationship between HCC and the expression of GRP75and HSP90. The results suggest that the expression of GRP75and HSP90in HCC is higher than non-tumor, which were further evidenced in HCC cells. Besides, along with the increased clinical stage of HCC, the expression of GRP75and HSP90is much higher.To determine whether combined use of GRP75(glucose-regulated protein75) inhibitor MKT-077and HSP90inhibitor17-AAG synergistically improves therapeutic efficacy in HCC in vitro and in vivo. Human HCC cell lines Bel-7402, HuH7, Hep3B (p53null) were exposed to vehicle, MKT-077or17-AAG alone or combinations of MKT-077and17-AAG. Cell viability, cell apoptosis, and subcellular localization of p53were analyzed by cell counting assays, flow cytometry and immunofluorescence staining, respectively. Expression levels of p53target genes were quantified using real-time PCR. A murine Bel-7402subcutaneous injection model was used to determine in vivo efficacy of MKT-077and17-AGG. GRP75and HSP90were found to be overexpressed in the majority of63human HCC tissues and expression correlated with advanced tumor staging. Inhibition of HSP90in HCC cells reduced levels of Akt, but increased levels of p53and GRP75. In addition,17-AAG was found to enhance binding of GRP75and p53, resulting in the retention of p53in the cytoplasm. Blocking GRP75with MKT-077potentiated the anti-tumor effects of17-AAG. MKT-077blocked the formation of GRP75-p53complexes, thereby facilitating translocation of p53into the nuclei and the induction of apoptosis-related genes. Finally, dual inhibition of HSP90and GRP75was found to significantly inhibit tumor growth in a liver cancer xenograft model. The GRP75inhibitor MKT-077enhances17-AAG-induced apoptosis in HCCs and increases p53-mediated inhibition of tumor growth in vivo. Dual targeting of GRP75and HSP90may be a useful strategy for the treatment of HCCs.Part Ⅱ Studing on the protective role of GRP75in PC12cells upon ionizing radiation injuryPrevious studies have shown that the expression of GRP75is upregulated by a low dose of ionizing radiation (IR). To evaluate the protective role of GRP75on cell proliferation in response to IR, GRP75was over-expressed in a rat adrenal pheochromocytoma cell line (PC12). It was revealed that GRP75overexpression desensitized PC12cells to IR-mediated cell injury. In addition, the expression of topoisomerase Ⅱα (Topo Ⅱα) was downregulated in PC12cells following y-ray IR. The effect of GRP75overexpression on Topo Ⅱα expression was examined. It was revealed that GRP75overexpression reversed the inhibitory effect of IR on Topo Ila expression. In conclusion, the data indicated that GRP75overexpression attenuates IR-induced injury in PC12cells by maintaining the expression of Topo Ⅱα.Our work found SalB and CP can upregulate the expressing of GRP75, then we investigate the effect of SalB and CP to PC12cell exposed to IR. We found SalB and CP can protect PC12cells from the IR injury by maintaining the proliferation of cells, besides, the expression of GRP75and Topo Ⅱα is unregulated. Only SalB inhibit the release of Reactive oxygen species. In conclusion, GRP75can protect cells from IR by inhibiting the release of ROS, keeping the expression of Topo Ⅱα which is important for cell proliferation.